Research Council Presentation UC San Diego Health Sciences March 5, 2014

1. “Quantifying the Time Progression of the Interaction of the Human Immune System with the Gut Microbiome” Research Council Presentation UC San Diego Health Sciences March 5, 2014 Dr. Larry Smarr Director, California Institute for Telecommunications and Information Technology Harry E. Gruber Professor, Dept. of Computer Science and Engineering Jacobs School of Engineering, UCSD http://lsmarr.calit2.net

2. Visualizing Time Series of 150 LS Blood and Stool Variables, Each Over 5-10 Years Calit2 64 megapixel VROOM

3. Only One of My Blood Measurements Was Far Out of Range--Indicating Chronic Inflammation 27x Upper Limit Episodic Peaks in Inflammation Followed by Spontaneous Drops Antibiotics Normal Range <1 mg/L Antibiotics Normal Complex Reactive Protein (CRP) is a Blood Biomarker for Detecting Presence of Inflammation

4. Adding Stool Tests RevealedOscillatory Behavior in an Immune Variable 124x Upper Limit Typical Lactoferrin Value for Active IBD Hypothesis: Lactoferrin Oscillations Coupled to Relative Abundance of Microbes that Require Iron Normal Range <7.3 µg/mL Lactoferrin is a Protein Shed from Neutrophils - An Antibacterial that Sequesters Iron

5. Colonoscopy Images Show Inflamed Pseudopolyps in 6 inches of Sigmoid Colon May 2011 Dec 2010

6. Confirming the Colonic Crohn’s Hypothesis:Finding the “Smoking Gun” with MRI Imaging “Long segment wall thickening in the proximal and mid portions of the sigmoid colon, extending over a segment of ~16 cm, with suggestion of intramural sinus tracts. Edema in the sigmoid mesentery and engorgement of the regional vasa recta.” – MRI report, Cynthia Santillan, M.D. UCSD Jan 2012 Crohn's disease affects the thickness of the intestinal wall. Having Crohn's disease that affects your colon increases your risk of colon cancer. Clinical MRI Slice Program Reveals Inflammation in 6 Inches of Sigmoid ColonThickness 15cm – 5x Normal Thickness

7. To Map Out the Dynamics of My Microbiome Ecology I Partnered with the J. Craig Venter Institute Illumina HiSeq 2000 at JCVI • JCVI Did Metagenomic Sequencing on Six of My Stool Samples Over 1.5 Years • Sequencing on Illumina HiSeq 2000 • Generates 100bp Reads • Run Takes ~14 Days • My 6 Samples Produced • 190.2 Gbp of Data • JCVI Lab Manager, Genomic Medicine • Manolito Torralba • IRB PI Karen Nelson • President JCVI Manolito Torralba, JCVI Karen Nelson, JCVI

8. We Downloaded Additional Phenotypes from NIH HMP For Comparative Analysis Download Raw Reads ~100M Per Person “Healthy” Individuals IBD Patients 2 Ulcerative Colitis Patients, 6 Points in Time 35 Subjects 1 Point in Time Larry Smarr 6 Points in Time 5 Ileal Crohn’s Patients, 3 Points in Time Total of 5 Billion Reads Source: Jerry Sheehan, Calit2 Weizhong Li, Sitao Wu, CRBS, UCSD

9. We Created a Reference DatabaseOf Known Gut Genomes Now to Align Our 5 Billion Reads Against the Reference Database • NCBI April 2013 • 2471 Complete + 5543 Draft Bacteria & Archaea Genomes • 2399 Complete Virus Genomes • 26 Complete Fungi Genomes • 309 HMP Eukaryote Reference Genomes • Total 10,741 genomes, ~30 GB of sequences Source: Weizhong Li, Sitao Wu, CRBS, UCSD

10. Computational NextGen Sequencing Pipeline:From “Big Equations” to “Big Data” Computing PI: (Weizhong Li, CRBS, UCSD): NIH R01HG005978 (2010-2013, $1.1M)

11. We Used SDSC’s Gordon Data-Intensive Supercomputer to Analyze a Wide Range of Gut Microbiomes Enabled by a Grant of Time on Gordon from SDSC Director Mike Norman • ~180,000 Core-Hrs on Gordon • KEGG function annotation: 90,000 hrs • Mapping: 36,000 hrs • Used 16 Cores/Node and up to 50 nodes • Duplicates removal: 18,000 hrs • Assembly: 18,000 hrs • Other: 18,000 hrs • Gordon RAM Required • 64GB RAM for Reference DB • 192GB RAM for Assembly • Gordon Disk Required • Ultra-Fast Disk Holds Ref DB for All Nodes • 8TB for All Subjects

12. We Recently Used Dell’s Supercomputer to Analyze An Additional 219 HMP and 110 MetaHIT Samples • Dell’s Sanger cluster • 32 nodes, 512 cores, • 48GB RAM per node • 50GB SSD local drive, 390TB Lustre file system • We used faster but less sensitive method with a smaller reference DB (duo to available 48GB RAM) • Only processed to taxonomy mapping • ~35,000 Core-Hrs on Dell’s Sanger • 30 TB data Source: Weizhong Li, UCSD

13. Using Scalable Visualization Allows Comparison of the Relative Abundance of 200 Microbe Species Comparing 3 LS Time Snapshots (Left) with Healthy, Crohn’s, UC (Right Top to Bottom) Calit2 VROOM-FuturePatient Expedition

14. Lessons From Ecological Dynamics:Invasive Species Dominate After Major Species Destroyed  ”In many areas following these burns invasive species are able to establish themselves, crowding out native species.” Source: Ponderosa Pine Fire Ecology http://cpluhna.nau.edu/Biota/ponderosafire.htm

15. Almost All Abundant Species (≥1%) in Healthy SubjectsAre Severely Depleted in Larry’s Gut Microbiome

16. Top 20 Most Abundant Microbial SpeciesIn LS vs. Average Healthy Subject Number Above LS Blue Bar is Multiple of LS Abundance Compared to Average Healthy Abundance Per Species 152x 765x 148x 849x 483x 220x 201x 169x 522x Source: Sequencing JCVI; Analysis Weizhong Li, UCSD LS December 28, 2011 Stool Sample

17. Comparing Changes in Gut Microbiome Ecology with Oscillations of the Innate and Adaptive Immune System LS Data from Yourfuturehealth.com Lysozyme & SIgA From Stool Tests Innate Immune System Therapy: 1 Month Antibiotics +2 Month Prednisone Time Points of Metagenomic Sequencing of LS Stool Samples Normal Adaptive Immune System Normal

18. Time Series Reveals Autoimmune Dynamics of Gut Microbiome by Phyla Therapy Six Metagenomic Time Samples Over 16 Months

19. “Arthur et al. provide evidence that inflammation alters the intestinal microbiota by favouring the proliferation of genotoxic commensals, and that the Escherichia coli genotoxin colibactin promotes colorectal cancer (CRC).” Christina Tobin Kåhrström Associate Editor, Nature Reviews Microbiology

20. Does Intestinal Inflammation Select for Pathogenic Strains That Can Induce Further Damage? AIEC LF82 “Adherent-invasive E. coli (AIEC) are isolated more commonly from the intestinal mucosa of individuals with Crohn’s disease than from healthy controls.” “Thus, the mechanisms leading to dysbiosis might also select for intestinal colonization with more harmful members of the Enterobacteriaceae*—such as AIEC—thereby exacerbating inflammation and interfering with its resolution.” E. coli/Shigella Phylogenetic Tree Miquel, et al. PLOS ONE, v. 5, p. 1-16 (2010) Sebastian E. Winter , et al., EMBO reports VOL 14, p. 319-327 (2013) *Family Containing E. coli

21. Chronic Inflammation Can Accumulate Cancer-Causing Bacteria in the Human Gut Escherichia coli Strain NC101

22. Deep Metagenomic Sequencing Enables Strain Analysis B2 D E B1 A Phylogenetic Tree 778 Ecoli strains =6x our 2012 Set S

23. We Divided the 778 E. coli Strains into 40 Groups, Each of Which Had 80% Identical Genes Group 0: D Group 5: B2 Group 26: B2 Group 7: B2 Group 2: E NC101 LF82 Group 4: B1 Group 3: A, B1 Group 9: S Group 18,19,20: S LS003 LS001 Median HE Median CD LS002 Median UC

24. Reduction in E. coli Over TimeWith Major Shifts in Strain Abundance Therapy Strains >0.5% Included

25. Thanks to Our Great Team! UCSD Metagenomics Team Weizhong Li Sitao Wu Calit2@UCSD Future Patient Team Jerry Sheehan Tom DeFanti Kevin Patrick Jurgen Schulze Andrew Prudhomme Philip Weber Fred Raab Joe Keefe Ernesto Ramirez JCVI Team Karen Nelson Shibu Yooseph Manolito Torralba SDSC Team Michael Norman Mahidhar Tatineni Robert Sinkovits UCSD Health Sciences Team William J. Sandborn Elisabeth Evans John Chang Brigid Boland David Brenner